1. Field of the Invention
The present invention relates to a treatment for myocardial infarction and blood clots within a patient, and more specifically to a therapy which enhances clot lysis comprising administering to a patient an antibody directed to xcex12-antiplasmin crosslinked to fibrin (xcex12AP-FX) which does not inhibit plasma xcex12-antiplasmin (xcex12AP). The invention also relates to a treatment for enhancing clot lysis comprising administering an antibody directed toward xcex12-antiplasmin crosslinked to fibrin which does not inhibit plasma xcex12AP together with a thrombolytic agent.
2. Background of the Invention
The initiating event of many myocardial infarctions (heart attacks) is the hemorrhage into atherosclerotic plaque. Such hemorrhage often results in the formation of a thrombus (or blood clot) in the coronary artery which supplies the infarct zone (i.e., an area of coagulation necrosis which results from an obstruction of blood circulation). This thrombus is composed of a combination of fibrin and blood platelets. The formation of a fibrin-platelet clot has serious clinical ramifications. The degree and duration of the occlusion caused by the fibrin-platelet clot determines the mass of the infarct zone and the extent of damage.
A. Treatment for Myocardial Infraction
The primary goal of current treatment for myocardial infarction involves the rapid dissolution of the occluding thrombus and the restoration of blood flow (xe2x80x9creperfusionxe2x80x9d). An agent which is capable of selectively binding to and affecting may enhance thrombolysis and may decrease the risk of general hemorrhage to the patient. A successful therapy must be capable of sustained effect so that reformation of the clot does not occur after the cessation of therapy. If the fibrin-platelet clot is able to reform, then the affected artery may become reoccluded.
The formation of fibrin-platelet clots in other parts of the circulatory system may be partially prevented through the use of anti-coagulants (such as heparin). Unfortunately, heparin has not been found to be universally effective in preventing reocclusion in myocardial infarction victims in which the degree of blood vessel occlusion (the degree of xe2x80x9cstenosisxe2x80x9d) is greater than or equal to 70%, particularly in those patients with severe residual coronary stenosis.
If an individual has formed a fibrin-platelet clot prior to the availability of medical assistance, the clot may be dissolved through the use of thrombolytic agents. A thrombolytic agent is a medicament capable of lysing the fibrin-platelet thrombus, and thereby permitting blood to again flow through the affected blood vessel. Such agents include, but are not limited to, streptokinase, prourokinase, urokinase, staphylokinase and tissue-type plasminogen activator (Ganz, W. et al., J. Amer. Coll. Cardiol. 1:1247-1253 (1983); Rentrop, K. P. et al., Amer. J. Cardiol. 54:29E-31E (1984); Gold, H. K. et al., Amer. J. Cardiol. 53:122C-125C (1984)).
B. Mechanism of Fibrin Clot Formation
Clots are composed of both fibrin and blood platelets in various ratios. The fundamental reaction in blood clotting involves the conversion of a soluble plasma protein (fibrinogen) into insoluble fibrin. The conversion of fibrinogen into fibrin is catalyzed by the enzyme, thrombin, which is a serine protease. Fibrin chains are crosslinked to each other by activated Factor XIII. Similarly activated Factor XIII crosslinks xcex12-AP to fibrin, concentrating the inhibitor on the clot surface. These two crosslinking events render the fibrin clot highly resistant to lysis. The general mechanism of blood clot formation is reviewed by Ganong, W. F. (In: Review of Medical Physiology, 9 ed., Lange, Los Altos, Calif., pp. 411-414 (1979)). Platelets are disk-shaped structures present in blood. They contribute to clot formation by both their incorporation with fibrin into an insoluble mass and by their enhancement of the rate of fibrinogen to fibrin conversion and by providing Factor XIII to enhance fibrin/xcex12-AP crosslinking. Platelets contribute to clot formation in myocardial infarction and are a major component of clots that reocclude coronary arteries that have been reperfused by treatment with a thrombolytic agent.
C. Mechanism of Clot Lysis and Natural Inhibition Thereof
Clot lysis is mediated by plasmin in vivo. Under natural conditions, plasminogen is converted to plasmin by tissue plasminogen activator (t-PA). Activation occurs on the fibrin surface, thus confining proteolytic activity to the appropriate site. After plasmin is set free into the circulation, it is rapidly combined with natural inhibitors. Inactivation of plasmin is the final and necessary step in the process of protecting against undesirable proteolysis. Such plasmin inhibitors include xcex12-antiplasmin, xcex12 -macroglobulin and xcex11-antitrypsin, all glycoproteins. xcex12-antiplasmin has a much higher affinity for plasmin than xcex12-macroglobulin and binds specifically to plasmin in a 1:1 ratio. The larger pool of xcex1-macroglobulin acts as a reservoir inhibitor. Kane, K. K., Ann. Clin. Lab. Sci. 14:443-449 (1984). Thus, clot lysis by the administration of plasminogen activators is limited by the rapid and irreversible inactivation of plasmin by plasmin inhibitors.
xcex12-antiplasmin has three functional domains: the reactive site for plasmin, the plasmin(ogen) or LBS-binding site [complementary to the LBS (lysine-binding site) of plasmin(ogen)], and the cross-linking site for fibrin. Mimuro, J. et al., Blood 69:446-453 (1987). Mimuro et al. disclose antibodies to xcex12-antiplasmin, one of which (JPTI-1) was specific to the reactive site of xcex12-antiplasmin and prevented formation of xcex12-antiplasmin-plasmin complexes, thereby inhibiting antiplasmin activity. However, Mimuro et al. do not teach administration of the JPTI-1 antibody to enhance clot lysis. Other antibodies specific for xcex12-antiplasmin are taught by Plow, E. F. et al., J. Biol. Chem. 255:2902-2906 (1980); Wimen, B. et al., Scan. J. Clin. Lab. Invest. 43:27-33 (1983); Hattey, E. et al., Thromb. Res. 45:485-495 (1987); Collen, U.S. Pat. No. 4,346,029 (1980); and Collen, U.S. Pat. No. 4,198,335 (1980).
D. xcex12-Antiplasmin Crosslinked to Fibrin
During clotting, xcex12AP is crosslinked to fibrin by Factor XIIIa. Aoki and colleagues have demonstrated that this crosslinking of xcex12AP to fibrin is important in preventing the xe2x80x9cendogenous fibrinolysisxe2x80x9d that occurs when fibrin-bound plasminogen is activated by fibrin-bound, endogenous, plasminogen activator (Aoki, N. et al., Blood 62:1118-1122 (1983)). This crosslinking may serve as a means of concentrating xcex12AP at the alpha chain site where fibrin appears particularly vulnerable to attach by plasmin (Pizzo, S. V. et al., J. Biol. Chem. 248:4574-4583 (1973)). Plasma clots deficient in crosslinked xcex12AP undergo a spontaneous lysis when suspended in buffer or plasma containing normal amounts of xcex12AP; the rate of lysis is proportional to the amount of xcex12AP incorporated into the clot (Sakata, Y. and Aoki, N., J. Clin. Invest. 69:536-542 (1982)). In a similar fashion, the crosslinking of xcex12AP to fibrin may be inhibited by a 0.13 mM concentration of a peptide that represents the 12 amino-terminal residues of xcex12AP (Kimura, S. et al., Blood 66:157-160 (1985)). This inhibition of crosslinking results in clots that lyse more readily upon exposure to fibrinolytic agents. In thrombolytic situations, fibrin-bound xcex12AP may be the most important inhibitor of clot lysis; whereas the chief role of soluble xcex12AP may be to prevent circulating plasmin from degrading other clotting factors.
Work with antibody RWR, has confirmed the importance of xcex12AP crosslinking in stabilizing the clot against lysis (Reed et al., Proc. Natl. Acad. Sci. USA 87:1114-1118 (1990), Reed et al., Circulation 82:164-168 (1990)). For example, in experiments using compressed and washed plasma clots (to clear away unbound xcex12AP), RWR alone causes the clots to undergo spontaneous lysis (Reed et al., Proc. Natl. Acad. Sci. USA 87:1114-1118 (1990). This spontaneous lysis is probably due to uninhibited plasmin generated by fibrin-associated t-PA""s action on fibrin bound plasminogen as has been suggested for xcex12AP deficiency (Aoki, N. et al., Blood 62:1118-1122 (1983)). In crosslinking to fibrin, xcex12AP forms a new epitope that is unique to clots.
An antibody that exclusively inhibits xcex12AP crosslinked to fibrin can be used to target plasminogen activators to a clot as well as to amplify their thrombolytic effects. Since antiplasmin crosslinked to fibrin is present in small quantities and only at the clot surface, such an antibody is the ideal inhibitor: it prolongs the half like of plasmin proximity to the clot while not interfering with the inactivation of circulating plasmin by soluble xcex12AP. As such it could not cause systemic thrombolysis. Such an agent can induce an ultra-specific xcex12AP deficiency at the site of the clot. In doing so, it could augment the normal clot lysis initiated by endogenous t-PA, and reproduce the spontaneous thrombolysis noted in studies of xcex12AP deficiency. If the antibody were given with a clot-specific agent such as t-PA, even more specific lysis would be obtained than has previously been seen. In addition, because of its combined specificity for the clot, and its capacity to simultaneously inhibit fibrin-crosslinked xcex12AP, this antibody would be extremely useful as a means of targeting plasminogen activators to the clot. Accordingly, the present invention provides antibodies which bind to and inhibit xcex12AP crosslinked to fibrin, but do not inhibit soluble xcex12AP.
E. Summary
In summary, a substantial goal of therapies aimed at treating myocardial infarction involves limiting necrosis by permitting early reperfusion and by preventing reocclusion. At present, this goal is partially achieved through the administration of thrombolytic agents capable of dissolving the potentially life-threatening fibrin-platelet clots. The potential benefit of employing such agents is, however, significantly offset by their lack of fibrin specificity (as in the case of streptokinase and urokinase), or by their relatively short biological half-life caused by plasmin inhibitors (which may result in reformation of the fibrin clot, and the accompanying reocclusion of the affected blood vessels). Hence, a need exists for an improvement in thrombolytic therapy which specifically enhances clot lysis, while minimizing fibrinogen breakdown and preventing reocclusion of the affected coronary artery.
It is a general object of the invention to provide a method for treating myocardial infarction or a blood clot within a patient.
It is a specific object of the invention to provide a method for treating myocardial infarction or a blood clot within a patient comprising administering to the patient a therapeutically effective amount of an antibody or fragment thereof capable of binding xcex12-antiplasmin crosslinked to fibrin wherein the antibody does not inhibit plasma xcex12-antiplasmin.
It is another specific object of the invention to provide a method of treatment for myocardial infarction or blood clots within a patient which comprises co-administering to the patient:
(a) an antibody or fragment thereof capable of binding xcex1c2-antiplasmin crosslinked to fibrin in a therapeutically effective amount wherein the antibody does not inhibit plasma xcex12-antiplasmin; and
(b) a thrombolytic agent in an amount sufficient to either (i) dissolve a fibrin-platelet clot or (ii) inhibit the formation of a fibrin-platelet clot.
It is a further specific object of the invention to provide a monoclonal antibody or fragment thereof wherein the antibody or fragment thereof is capable of binding xcex12-antiplasmin crosslinked to fibrin and does not inhibit plasma xcex12-antiplasmin.
It is another specific object of the invention to provide a kit useful for carrying out the above-described methods, comprising a carrier means being compartmentilized in close confinement to receive two or more container means therein, which comprises:
(1) a first container means containing a therapeutically effective amount of the above-described antibody or fragment thereof; and
(2) a second container containing a therapeutically effective amount of a thrombolytic agent.
Further objects and advantages of the present invention will be clear from the description that follows.